Project Summary It has been demonstrated recently that a diverse set of enzyme-mediated modifications are found internally within RNAs which markedly influence the fate of RNAs in cells. Most of the RNA modifications are installed and removed by enzymes, termed writers (e.g., methyltransferases) and erasers (e.g., demethylases). Importantly, studies to date have focused solely on individual modifications on isolated RNA. However, recent data from my laboratory and others? suggest cross-talk between modifications or the modifying enzymes influence both the depositions and consequences of several RNA modifications. Our preliminary data suggest that the level of one type of mRNA modification influences the level of the other, indicating that mRNA modifications may function in a previously undetermined combinatorial fashion. We also discovered that the level of a tRNA modification is concurrently regulated with a mRNA modification by a complex of RNA modifying enzymes. These data suggest the coordination between RNA modifying enzymes. However, the detailed mechanism of how these RNA modifying enzymes are coordinated and the biological function of the interplay of RNA modifications in tRNA and mRNA is not known. We discovered that a rRNA methyltransferase has dual enzymatic activities and install di-methylation on both rRNA and mRNA. Although the rRNA methyltransferase works equally well on rRNA and mRNA in vitro, the levels of mRNA modification are much lower in cells, suggesting that substrate preference is regulated. However, it is not known how this rRNA methyltransferase is controlled to achieve the substrate selectivity. To study whether a combinatorial modification code exists in mRNA, we will study the interplay between m3C and m6A in mRNA. Specifically, we will investigate whether the deposition of m3C affects m6A, and vice versa. We will also study the possible mechanism of the combinatorial mRNA modification in the recruitment of modification binding proteins, catalysis of RNA modifying enzymes, and the accessibility of the substrate. To understand the potential correlation between mRNA and tRNA through RNA modifications, we will investigate the interactions and the cellular consequence of the disturbed interactions of a protein complex of Trmt10A and YTHDF2. To understand how a dimethyladenosine methyltransferase regulates rRNA and mRNA modification, we will investigate the functions of dimethyladenosine in rRNA and mRNA, respectively. And then to study the regulatory mechanism by which this enzyme achieves substrate preference. Collectively, these innovative studies will provide fundamental insights into how multiple types of enzyme-mediated RNA modifications synergistically function.